Isolated microenvironments like the synapse exist throughout the nervous system where the concentration of ions is regulated by accessory cells, including glia, quite independently from the surrounding tissues. The ionic composition of these microenvironments is key for neuronal function. Despite the fact that glial regulation of ion concentration in microenvironments is a main mode for regulating neuronal activity, our understanding of this type of regulation by glia is limited, especially for ions like Cl- and HCO3-. Furthermore, models are lacking where a comprehensive analysis can be performed on the glial ion channels and transporters involved in regulating ion concentrations, and how these proteins impact neuronal output, from molecule to animal behavior. In our over 10 years of effort aimed at advancing understanding of glia-neurons interaction and its impact on animal behavior using the model C. elegans, we have recently taken the unbiased approach of sequencing the mRNA of Amphid sheath glia. In this application, we propose to establish the mechanism by which one of the identified enriched genes, the glial Cl-/HCO3- permeable channel CLH-1, regulates neuronal output and animal behavior. We previously published that CLH-1 mediates pH regulation in the worm nervous system. Our preliminary results now show that CLH-1 is needed for normal nose-touch behavior. We hypothesize that glial CLH-1 regulates the activity of touch neurons via a direct effect of the permeating ions Cl- and HCO3- on neuronal DEG-1 channels. Thus, the specific aims of this application are: 1) In neurons, to establish the mechanism of neuronal dysfunction when clh-1 is knocked-out, 2) In glia, to determine whether it is the loss of permeation of Cl-, HCO3-, or both that produces the phenotype of clh-1 knock-out worms. Furthermore, in aim 3 we will exploit our proven approach to identify additional glial ion channels and transporter genes that are critical for the glial control of neuronal function and animal behavior: 3) To identify novel glial ion channels and transporters involved in glia-neurons interaction. The importance of regulating ion concentrations in neuronal microenvironments is underscored by the fact that several neurological diseases such as deafness, epilepsy, Alzheimer's, and even demyelinating diseases like multiple sclerosis are characterized by loss of ionic homeostasis. We propose here to use methodologies we have developed and proven to be effective to test mechanisms by which dysregulation of Cl- and HCO3- homeostasis in C. elegans leads to severe neuronal pathology. In addition, we will test the involvement of newly identified genes encoding glial channels and transporters in glia-neurons interaction.